Significant evidence exists suggesting that NHE-3, an Na/H antiporter isoform, encodes a significant fraction of proximal tubule apical membrane Na/H antiporter activity. The overall aim of the present studies is to examine the molecular mechanisms responsible for regulation of NHE-3. In Aim l, studies will examine chronic regulation of NHE-3 in three settings, chronic acidosis, chronic increases in tonicity, and chronic increases in cell cAMP levels. The majority of the studies will be performed in cultured cells, and will examine whether effects on NHE-3 activity in these models are mediated by: 1) changes in mRNA abundance due to either changes in transcription rate or mRNA stability, 2) changes in protein abundance due to changes in synthesis rate or stability, 3) cellular trafficking, 4) posttranslational modification of NHE-3; or 5) regulation of a binding protein. Where appropriate, studies will he performed in vivo in rats to determine whether findings in cell culture apply. In preliminary studies, we have demonstrated that chronic acidosis activates c-src, and inhibition of src family nonreceptor tyrosine kinases prevents acid-induced activation of NHE-3. In Aim 2, studies will address the molecular mechanisms by which c-src is activated, and the role of c- src in the regulation of renal acidification. Specifically, studies will examine whether the effect of pH on c-src is specific for proximal tubule cells or occurs in other cells, and whether the effect is specific for c- src or also involves other src family nonreceptor tyrosine kinases. Studies will then examine how c-src is modified by acid with respect to phosphorylation, binding, and localization within cells. Studies will address whether overexpression of a constitutively activated c-src increases Na/H antiporter activity. Using mice in which the c-src gene or the c-yes gene has been disrupted, we will examine the role of these kinases in the regulation of renal acidification in vivo. Lastly, studies will address whether c-src plays a role in acid-induced immediate early gene activation and in hormonal activation of the Na/H antiporter. The last aim will address the molecular mechanisms by which angiotensin II and endothelin regulate NHE-3 activity. Mechanisms addressed will focus on trafficking and posttranslational modification. Studies will also measure ambient endothelin levels in the proximal tubule. Lastly, mice in which the genes for ET-3 or ETB have been disrupted, will be used to study the role of this pathway in the regulation of renal function.